The general focus of our research is on the process of differentiation. In particular, the coordination of the numerous cellular and molecular events that must occur as proliferating cells transition to terminal differentiation. Two key events in this transition are cell cycle exit and tissue specific gene expression. The lack of coordination between cell proliferation and terminal differentiation underlies the molecular mechanisms of cancer. An attractive model to study these processes is osteoblast differentiation. These cells differentiate in a controlled multi step program with distinct proliferative and postproliferative stages. Our previous studies on the role of the retinoblastoma protein and p300/CBP in the regulation of osteoblast differentiation revealed a link between osteopontin expression and the enzymatic activity of alkaline phosphatase, which creates inorganic phosphate in the medium. The elevation of free phosphate is sufficient to signal the induction of osteopontin RNA and protein. The discovery that inorganic phosphate in the medium results in increased expression of osteopontin not only in MC3T3-E1 cells but also in other cell types including the fibroblast cell line NIH3T3, defines a novel mechanism for gene regulation. Osteopontin is a multi-faceted extracellular matrix protein expressed in most tissues. Studies involving reduced levels of osteopontin have shown that it is required for bone remodeling, wound healing and as a key cytokine in efficient type-1 immune responses. Elevated levels of the osteopontin are associated with various disease states including transformation, metastasis, atherosclerosis and kidney disease. A high level of expression of osteopontin is associated with the poor prognosis of many cancers. Although expression of this gene has been demonstrated in multiple tissue types its regulation is not well understood. We are currently defining the signal transduction pathway(s), promoter element(s) and transcription factor(s) that regulate osteopontin expression in response to elevated phosphate. The possibility that inorganic phosphate can act as a signaling molecule capable of altering gene expression in osteoblast development prompted us to conduct a microarray analysis of phosphate treated osteoblasts. The array analysis identified numerous genes both positively and negatively regulated by increased phosphate concentrations. Nrf2 is one such upregulated gene. Nrf2 is the key transcription factor in the regulation of phase II detoxifying enzymes, responsible for the detoxification of carcinogens. Loss of function of Nrf2 has been linked to carcinogenesis. This line of study has lead to novel insights into the transcriptional and post-translational regulation of Nrf2. We have determined that protein synthesis is not required for an increase in RNA, and cloning of the promoter confirms that Nrf2 is regulated transcriptionally. Although Nrf2 has been mainly associated with the cellular response to chemopreventative agents, our data suggests it may play important roles in varied cellular processes. This data also implicates Nrf2 as an important transcription factor in the later stages of osteogenesis and may be the mechanism by which osteoblasts survive the harsh environment created during bone formation. This analysis has also lead to the discovery that elevated levels of inorganic phosphate are capable of regulating a number of genes and may represent a globally important signaling molecule. These data will be useful in further understanding the critical interactions between osteoblasts and their extracellular environment as differentiation proceeds. A related project in the lab revolves around the role of p270 in differentiation. p270 is an integral member of human SWI/SNF complexes. SWI/SNF complexes are chromatin modifying machines that play fundamental roles in the regulation of gene expression during cell growth and development in all organisms. Sequence analysis revealed that p270 has a group of four LXXLL (L is leucine and X is any amino acid) motifs located towards the C-terminus of the protein. This motif has been demonstrated to be necessary and sufficient for interaction with liganded nuclear hormone receptors. The function of other LXXLL containing proteins is to assist in the formation of a complex between upstream transcriptional activators/enhancers and the basal transcriptional machinery, including TBP. The potential interaction between various nuclear hormone receptors and p270, a protein associated with a complex that contains chromatin-modifying activity, is very exciting. It suggests a role for p270 as an integrator of hormone signaling and transcription initiation. We are currently using a model of adipocyte differentiation to examine the potential role(s) of p270 and the SWI/SNF complex in differentiation.